Spatially-addressable immobilization of anti-ligands on surfaces

ABSTRACT

Methods and compositions are described for immobilizing anti-ligands, such as antibodies or antigens, hormones or hormone receptors, oligonucleotides, and polysaccharides on surfaces of solid substrates for various uses. The methods provide surfaces covered with caged binding members which comprise protecting groups capable of being removed upon application of a suitable energy source. Spatially addressed irradiation of predefined regions on the surface permits immobilization of anti-ligands at the activated regions on the surface. Cycles of irradiation on different regions of the surface and immobilization of different anti-ligands allows formation of an immobilized matrix of anti-ligands at defined sites on the surface. The immobilized matrix of anti-ligands permits simultaneous screenings of a liquid sample for ligands having high affinities for certain anti-ligands of the matrix. A preferred embodiment of the invention involves attaching photoactivatable biotin derivatives to a surface. Photolytic activation of the biotin derivatives forms biotin analogs having strong binding affinity for avidin. Biotinylated anti-ligands can be immobilized on activated regions of the surface previously treated with avidin.

This application is a divisional of U.S. Ser. No. 07/612,671, filed Nov.13, 1990, now U.S. Pat. No. 5,252,743, which is a continuation-in-partof U.S. Ser. No. 07/435,316 filed Nov. 13, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods and compositionsuseful for immobilizing anti-ligands on surfaces. The immobilizedanti-ligands, which can be, for example, hormones or hormone receptors,antibodies or antigens, oligosaccharides, and oligonucleotides, may beused in a variety of screening and assay methodologies for ligands inliquid media.

Certain biological molecules are known to interact and bind to othermolecules in a very specific manner. Essentially any molecules having ahigh binding specificity for each other can be considered aligand/anti-ligand pair, e.g., a vitamin binding to a protein, acell-surface receptor binding to a hormone or drug, a glycoproteinserving to identify a particular cell to its neighbors, an IgG-classantibody binding to an antigenic determinant, an oligonucleotidesequence binding to its complementary fragment of RNA or DNA, and thelike.

The specific binding properties of anti-ligands for ligands haveimplications for many fields. For example, the strong binding affinityof antibodies for specific determinants on antigens is critical to thefield of immunodiagnostics. Additionally, pharmaceutical drug discovery,in many cases, involves discovering novel drugs having desirablepatterns of specificity for naturally-occurring receptors or otherbiologically important anti-ligands. Many other areas of research existin which the selective interaction of anti-ligands for ligands isimportant and are readily apparent to those skilled in the art.

The immobilization of anti-ligands onto surfaces is an important step inperforming repetitive assays and screenings of ligands with solid phasesystems. Previous methods of attaching anti-ligands to surfaces arelimited by low reaction efficiencies or by a general inability toregionally and selectively attach a plurality of anti-ligands to thesurface.

A large variety of methods are known for attaching biological moleculesto solid supports. See generally, Affinity Techniques. EnzymePurification: Part B. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby,M. Wilchek, Acad. Press, N.Y. (1974) and Immobilized Biochemicals andAffinity Chromatography, Advances in Experimental Medicine and Biology,vol. 42, ed. R. Dunlap, Plenum Press, N.Y. (1974), which areincorporated herein by reference. For example, U.S. Pat. No. 4,681,870describes a method for introducing free amino or carboxyl groups onto asilica matrix. These groups may subsequently be covalently linked to,e.g., a protein or other anti-ligand, in the presence of a carbodiimide.Alternatively, a silica matrix may be activated by treatment with acyanogen halide under alkaline conditions. The anti-ligand is covalentlyattached to the surface upon addition to the activated surface. Anotherexample is presented in U.S. Pat. No. 4,282,287, which describes amethod for modifying a polymer surface through the successiveapplication of multiple layers of biotin, avidin and extenders. Also,U.S. Pat. No. 4,762,881 describes a method for attaching a polypeptidechain to a solid substrate by incorporating a light-sensitive unnaturalamino acid group into the polypeptide chain and exposing the product tolow-energy ultraviolet light.

Similarly, a variety of techniques have been developed for attachingoligonucleotides to surfaces. For example, U.S. Pat. No. 4,542,102describes a method employing a photochemically active reagent (e.g., apsoralen compound) and a coupling agent, which attaches the photoreagentto the substrate. Photoactivation of the photoreagent binds a nucleicacid sequence to the substrate to give a surface-bound probe for acomplementary oligonucleotide of the sequence. However, this method haslow quantum yields in protic solvents, lacks spatial directability, andrelies upon initial affinity between the photoreagent and nucleic acidsprior to photoactivation.

U.S. Pat. No. 4,562,157 describes a technique for attaching biochemicalligands to surfaces by attachment of a photochemically reactive arylazide. Irradiation of the azide creates a reactive nitrene which reactsirreversibly with macromolecules in solution resulting in the formationof a covalent bond. The high reactivity of the nitrene intermediate,however, results in both low coupling efficiencies and many potentiallyunwanted products due to nonspecific reactions.

Thus, there exists a need for improved methods for attaching a broadrange of anti-ligands to predefined regions of a solid support surface.The methods should efficiently provide stable attachment of selectedanti-ligands to the activated surface regions, yet attachment should berestricted to the activated regions. The present invention fulfillsthese and other needs.

SUMMARY OF THE INVENTION

Novel methods and compositions of matter are provided for immobilizinganti-ligands on predefined regions of a surface of a solid support. Themethods involve attaching to the surface a caged binding member whichhas a relatively low affinity for other potentially binding species,such as anti-ligands and specific binding substances. The caged bindingmember is convertible, e.g., by irradiation, to a binding memberultimately capable of immobilizing a desired anti-ligand, preferably viaa non-covalent interaction. Predefined regions of the surface areselectively irradiated to convert the caged binding members in thepredefined regions to activated binding members. The desiredanti-ligands subsequently can be immobilized on the activated regions ofthe surface.

Importantly, the spatial addressability afforded by the method of thepresent invention allows the formation of patterned surfaces havingpreselected reactivities. For example, by using lithographic techniquesknown in the semiconductor industry, light can be directed to relativelysmall and precisely known locations on the surface. It is, therefore,possible to activate discrete, predetermined locations on the surfacefor attachment of anti-ligands. The resulting surface will have avariety of uses. For example, direct binding assays can be performed inwhich ligands can be simultaneously tested for affinity at differentanti-ligands attached to the surface. Ligand binding is detected by atechnique such as autoradiography when the ligand is radioactivelylabelled. Alternatively, fluorescence or other optical techniques can beused. By determining the locations and intensities of labels on thesurface it is possible to simultaneously screen ligands for affinity toa plurality of anti-ligands.

A further understanding of the nature and advantages of the inventionmay be realized by reference to the remaining portions of thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1d present chromatographic results showing that NVOC-biotin-ONPis converted to biotin-ONP upon illumination in solution.

FIG. 2 presents radioligand binding results showing NVOC-biotin-OMe haslow affinity for avidin prior to illumination but high affinity afterillumination in solution.

FIG. 3 presents gamma counting results showing that illumination ofmembrane-bound NVOC-biotin increases the binding of radioactive avidinto the membrane.

FIG. 4 presents fluorescence results showing the spatial immobilizationof Fluorescein-Streptavidin on a biotinylated surface.

FIG. 5 presents fluorescence results showing the spatial immobilizationof Fluorescein-Biotin on a surface modified with Streptavidin.

FIG. 6 presents fluorescence results showing the spatial immobilizationof Fluorescein-Streptavidin on a surface having biotin bound by apolyether linker.

FIG. 7 presents fluorescence results showing the spatial immobilizationof Fluorescein-Streptavidin on a biotinylated surface.

FIG. 8 presents fluorescence results showing the spatial immobilizationof Bodipy-Streptavidin on a biotinylated surface.

FIG. 9 presents fluorescence results showing the effect of insertinglinkers of different lengths on the binding of Fluorescein-Streptavidin.

FIG. 10a presents fluorescence results showing binding ofFluorescein-anti-Rabbit IgG to a slide having multiple anti-ligands.

FIG. 10b presents fluorescence results showing binding ofFluorescein-anti-Mouse IgG to a slide having multiple anti-ligands.

FIG. 10c presents fluorescence results showing binding of a mixture ofFluorescein-anti-Rabbit IgG and Fluorescein-anti-Mouse IgG to a slidehaving multiple anti-ligands.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

CONTENTS:

I. Glossary

II. Overview

III. Substrate Preparation

IV. Properties of Binding Members and Caging Groups

A. Caging Groups

B. Irradiation of Caged Compounds

V. Attachment of Anti-Ligands

VI. Screenings and Assays

VII. Examples

I. Glossary

The following terms have the following meanings and abbreviations asused herein:

1. Surface (S): A surface is any generally two-dimensional structure ona solid substrate. A surface may have steps, ridges, kinks, terraces andthe like without ceasing to be a surface.

2. Predefined Region (S_(i)): A predefined region is a localized area ona surface which is or is intended to be activated. The predefined regionmay have any convenient shape, e.g., circular, rectangular, elliptical,etc.

3. Crosslinking Group (X): A crosslinking group is a bifunctionalchemical entity that serves to connect a binding member to a surface.Usually, crosslinking groups will be heterobifunctional, i.e., they willhave different chemical reactivities on either end of the linking group.

4. Binding Member (B): A binding member is any substance having asufficiently high affinity for another substance. A binding member willhave a sufficiently high affinity for another substance for practice ofthis invention when it effectively binds the substance withoutirreversibly separating from it throughout the handling and performancesteps of the invention. A binding member is usually, but not always,connected to a surface via a crosslinking group.

5. Caged Binding Member (B*): A caged binding member is a binding memberthat is provided with a removable (labilizable) chemical protectinggroup. Such protecting groups are characterized by their abilities todeter effective binding between the binding member to which they areattached and other substances otherwise having affinity for the bindingmember. Also, the protecting groups are readily labilizable, i.e., theycan be detached from the binding member to which they are attached uponexposure to a suitable source of energy.

6. Specific Binding Substance (SBS): A specific binding substance is acompound having a sufficiently high affinity and selectivity for bindingto a binding member to permit practice of the present invention. Aspecific binding substance may be larger or smaller than the bindingmember to which it specifically binds. The specific binding substanceserves as a bridge for attaching an anti-ligand to binding members onthe surface.

7. Anti-ligand (AL_(i)): An anti-ligand is a molecule that has a knownor unknown affinity for a given ligand and can be immobilized on apredefined region of the surface. Anti-ligands may benaturally-occurring or manmade molecules. Also, they can be employed intheir unaltered state or as aggregates with other species. Anti-ligandsmay be reversibly attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. By"reversibly attached" is meant that the binding of the anti-ligand (orspecific binding member or ligand) is reversible and has, therefore, asubstantially non-zero reverse, or unbinding, rate. Such reversibleattachments can arise from noncovalent interactions, such aselectrostatic forces, van der Waals forces, hydrophobic (i.e., entropic)forces, and the like. Furthermore, reversible attachments also may arisefrom certain, but not all covalent bonding reactions. Examples include,but are not limited to, attachment by the formation of hemiacetals,hemiketals, imines, acetals, ketals, and the like (See, Morrison et al.,"Organic Chemistry", 2nd ed., ch. 19 (1966), which is incorporatedherein by reference). Examples of anti-ligands which can be employed bythis invention include, but are not restricted to, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as on viruses, cells or other materials),hormones, drugs, oligonucleotides, peptides, enzymes, substrates,cofactors, lectins, sugars, oligosaccharides, cells, cellular membranes,and organelles.

8. Ligand (L): A ligand is a solvated molecule that is recognized by aparticular anti-ligand. Examples of ligands that can be investigated bythis invention include, but are not restricted to agonists andantagonists for cell membrane receptors, toxins and venoms, viralepitopes, hormones (e.g., opiates, steroids etc.), hormone receptors,peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars,oligonucleotides, oligosaccharides, proteins, and monoclonal antibodies.

II. Overview

The present invention provides methods for forming predefined regions ona surface of a solid support, wherein the predefined regions are capableof immobilizing anti-ligands. The methods make use of caged bindingmembers attached to the surface to enable selective activation of thepredefined regions. The caged binding members are converted to bindingmembers ultimately capable of binding anti-ligands upon selectiveactivation of the predefined regions. The activated binding members arethen used to immobilize anti-ligands on the predefined region of thesurface. The above procedure can be repeated at the same or differentsites on the surface so as to provide a surface prepared with aplurality of regions on the surface containing the same or differentanti-ligands. When the anti-ligands have a particular affinity for oneor more ligands, screenings and assays for the ligands can be conductedin the regions of the surface containing the anti-ligands.

The present methods are distinguished by the employment of novel cagedbinding members attached to the substrate. Caged (unactivated) membershave a relatively low binding affinity for anti-ligands or specificbinding substances when compared with the corresponding affinities ofactivated binding members. Thus, the binding members are protected untila suitable source of energy is applied to the regions of the surfacedesired to be activated. Upon application of a suitable energy source,the caging groups labilize, thereby presenting the activated bindingmember. A typical energy source will be light.

Once the binding members on the surface are activated they may beattached to an anti-ligand. The anti-ligand chosen may be a monoclonalantibody, a nucleic acid sequence, a drug receptor, etc. The anti-ligandwill usually, though not always, be prepared so as to permit attachingit, directly or indirectly, to a binding member. For example, a specificbinding substance having a strong binding affinity for the bindingmember and a strong binding affinity for the anti-ligand may be used asa bridge. Alternatively, a covalently-linked conjugate of the specificbinding substance and anti-ligand may be used. The method uses ananti-ligand prepared such that the anti-ligand retains its activitytoward a particular ligand.

Preferably, the caged binding member attached to the solid substratewill be a photoactivatable biotin analog, i.e., a biotin molecule thathas been chemically modified with photoactivatable protecting groups sothat it has a significantly reduced binding affinity for avidin oravidin analogs compared to that of natural biotin. In a preferredembodiment, the protecting groups localized in a predefined region ofthe surface will be removed upon application of a suitable source ofradiation to give binding members, that are biotin or a functionallyanalogous compound having substantially the same binding affinity foravidin or avidin analogs as does biotin.

In another preferred embodiment,. avidin or an avidin analog will beincubated with activated binding members on the surface until the avidinbinds strongly to the binding members. The avidin so immobilized onpredefined regions of the surface, can then be incubated with a desiredanti-ligand or conjugate of a desired anti-ligand. The multiple biotinbinding sites on avidin allow simultaneous binding of biotin attached tothe surface and biotin attached to the anti-ligand. The anti-ligand willpreferably be biotinylated, e.g., a biotinylated antibody, when avidinis first immobilized on the predefined regions of the surface.Alternatively, a preferred embodiment will present anavidin/biotinylated anti-ligand complex, which has been previouslyprepared, to activated binding members on the surface.

The following equations depict the best modes of practicing theinvention:

The attachment of binding members (B) to a surface (S) of a solidsubstrate is illustrated by the following reactions:

    S+B*→S-B* or

    S+B→S-B                                             (1)

    S-B+*→S-B*                                          (2)

where "*" represents a protecting (caging) group. B* is a caged bindingmember. The protecting groups can either be attached to the bindingmembers once the binding members have been attached to the surface, ormore preferably, they will be attached to binding members prior toattaching the binding members to the surface.

Also, surface attachment of binding members can be effected through theuse of crosslinking groups (X). This is represented by the followingreactions:

    S+X→S -X                                            (1)

    S-X+B*→S-X-B*                                       (2) or

    S-X+B→S-X-B                                         (2')

    S-X-B+*→S-X-B*

The crosslinking groups will usually, though not always, beheterobifunctional chemical species having a first reactivity whichpermits the crosslinking group to bind readily to the surface and asecond reactivity which permits the crosslinking group to bind readilywith binding members.

Predefined regions (S_(i)) on the surface can be activated for ultimateimmobilization of anti-ligands in the predefined regions by selectivelyirradiating predefined regions to convert photoactivatable bindingmembers in the predefined region to binding members. This process isillustrated by the following reactions: ##STR1## The free protectinggroup, "*" may or may not undergo decomposition reactions. It willusually be washed from the surface, depending upon whether it interfereswith subsequent reactions.

Immobilization of anti-ligands (AL_(i)) on predefined regions of thesurface can be effected by binding the anti-ligands directly to bindingmembers or through a bridging specific binding substance (SBS). Thespecific binding substance may be introduced to binding members alone oras a previously prepared conjugate of the anti-ligand. Multipleanti-ligands may be immobilized on the surface when the specific bindingsubstance contains multiple binding sites. Also, it should be noted thatan advantage of using a specific binding substance is that animmobilization technique generic for many anti-ligands may be employed.Immobilization of anti-ligands on predefined regions of the surface isillustrated by the following reactions:

    S.sub.i -X-B+AL.sub.i →Si.sub.i -X-B-AL.sub.i or

    S.sub.i -X-B+SBS-AL.sub.i →Si.sub.i -X-B-SBS-AL.sub.i or

    S.sub.i -X-B+SBS→Si-X-B-SBS                         (1)

    Si-X-B-SBS+AL.sub.i →Si-X-B-SBS-AL.sub.i            (2),

where the horizontal lines B-SBS, B-AL_(i), or SBS-AL_(i) representbonding between two molecules, preferably a non-covalent bond.

An example of immobilizing a different anti-ligand (AL_(j)) on adifferent predefined region (S_(j)) of the surface is shown by theequation:

    S.sub.j -X-B+SBS-AL.sub.j →S.sub.j -X-B-SBS-AL.sub.j.

Repetition of the above steps on different regions of the surface canproduce a matrix of anti-ligands immobilized on the surface. Such amatrix can have any desired pattern of anti-ligands. An example of sucha matrix is given below:

    ______________________________________                                        AL.sub.i        AL.sub.j    B*                                                B*              B*          AL.sub.k                                          AL.sub.1        B*          B*.                                               ______________________________________                                    

An immobilized anti-ligand on a surface will have a specific bindingaffinity for a particular ligand (L). An example of a direct assay on apredefined region of the surface for the presence of a labeled ligand(L') in a liquid medium is illustrated by the following reaction:

    S.sub.i -X-B-SBS-AL.sub.i +L'→Si-X-B-SBS-AL.sub.i -L'.

The resulting surface can be washed free of unbound ligand and analyzedfor the presence of label. The labels will serve as markers localized atthe predefined regions on the surface corresponding to the presence ofanti-ligands for the ligand at those predefined regions.

Some examples of competitive assays, in which a target ligand (L)"competes" with another ligand (L') for a site on the surface, areillustrated by the following reactions:

    S.sub.i -X-B-SBS-AL.sub.i -L'+L→S.sub.i -X-B-SBS-AL.sub.i -L+L'(1).

    S.sub.i -X-B-SBS-AL.sub.i +L/L'→S.sub.i -X-B-SBS-AL.sub.i -L'+L (2).

The presence of target ligand can be determined by analyzingappropriately for the loss or buildup of label on the predefined regionsof the surface.

III. Substrate Preparation

Essentially, any conceivable solid substrate may be employed in theinvention. The substrate may be biological, nonbiological, organic,inorganic, or a combination of any of these, existing as particles,strands, precipitates, gels, sheets, tubing, spheres, containers,capillaries, pads, slices, films, plates, slides, etc. The substrate mayhave any convenient shape, such as a disc, square, sphere, circle, etc.The substrate and its surface preferably form a rigid support on whichto carry out the reactions described herein. The substrate and itssurface should also be chosen to provide appropriate light-absorbingcharacteristics. For instance, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a wide variety of polymers such as(poly) tetrafluoroethylene, (poly) vinyl idenedifluoride, orcombinations thereof. Other substrate materials will be readily apparentto those of skill in the art upon review of this disclosure. In apreferred embodiment the substrate is flat glass or single-crystalsilicon with surface features of less than 10 Å.

Surfaces on the solid substrate will usually, though not always, becomposed of the same material as the substrate. Thus, the surface may becomposed of any of a wide variety of materials, for example, polymers,plastics, resins, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, etc., provided only thatcaged binding members can be attached firmly to the surface of thesubstrate. Preferably, the surface will contain reactive groups, whichcould be carboxyl, amino, hydroxyl, or the like. Most preferably, thesurface will be optically transparent and will have surface Si-OHfunctionalities, such as are found on silica surfaces.

The surface of the substrate is preferably provided with a layer ofcrosslinking groups, although it will be understood that thecrosslinking groups are not required elements of the invention. Thecrosslinking groups are preferably of sufficient length to permitbinding members on the surface to interact freely with compounds insolution. Crosslinking groups may be selected from any suitable class ofcompounds, for example, aryl acetylenes, ethylene glycol oligomerscontaining 2-10 monomer units, diamines, diacids, amino acids, orcombinations thereof. Other crosslinking groups may be used in light ofthis disclosure.

Crosslinking groups may be attached to the surface by a variety ofmethods which are readily apparent to one having skill in the art. Forexample, crosslinking groups may be attached to the surface by siloxanebonds formed via reactions of crosslinking groups bearing trichlorosilylor trisalkoxy groups with hydroxyl groups on the surface of thesubstrate. Preferably, the crosslinking group used with a glass surfaceis N-BOC-aminopropyltriethoxy silane. The crosslinking groups mayoptionally be attached in an ordered array, i.e., as parts of the headgroups in a polymerized Langmuir Blodgett film. Clearly, the type ofcrosslinking group selected, and the method selected for attaching it tothe surface, will depend primarily on the crosslinking group havingsuitable reactivity with the binding member desired to be attached tothe surface.

Additional length may be added to the crosslinking groups by theaddition of single or multiple linking groups. Such linking groups arepreferably heterobifunctional, having one end adapted to react with thecrosslinking groups and the other end adapted to react with the bindingmember or an another linking group. The linking groups may be attachedby a variety of methods which are readily apparent to one skilled in theart for instance, esterification or amidation reactions of an activatedester of the linking group with a reactive hydroxyl or amine on the freeend of the crosslinking group. A preferred linking group isN-BOC-6-aminocaproic acid (i.e., N-BOC-6-aminohexanoic acid) attached bythe BOP-activated ester. After deprotection to liberate the free amineterminus, another N-BOC-aminocaproic linker can be added. Attachment ofcrosslinking and linking groups to caged binding members are discussedmore fully below.

Many methods are available for immobilizing the binding members of thepresent invention on surfaces. The binding members may be linked to thesurface in their active forms, and later provided with protecting(caging) groups. More preferably, binding members will be provided intheir protected forms. The method chosen for linking binding members tothe surface will depend upon the chemical properties of the bindingmember selected for attachment to the surface. A preferred method forimmobilizing the binding members of the present invention involveschemical derivatization or activation of the caged binding member priorto attachment to the surface or linker. This derivative or activatedspecies is then reacted with functionalities on the substrate to givethe desired linkage. For example, one method for attaching a bindingmember to a surface employs a heterobifunctional crosslinking reagent,such as diepoxide, which both activates the surface and provides a groupthat reacts with an activated binding member. Alternatively, the surfacecan be activated with cyanogen bromide. Reaction with a binding membercontaining a terminal amino group permits attachment of the bindingmember to the surface. (U.S. Pat. No. 4,542,102). In the presence of acarbodiimide or other activating agent, for example, the amine group canbe coupled to the carboxyl terminus of a binding member desired to beimmobilized on the surface.

A preferred embodiment of the present invention involves attaching"caged" derivatives of biotin or biotin analogs to a glass surface.Caged biotin may be attached to the surface through strong noncovalentinteractions, e.g., by crosslinking via a suitable linker to anotherbiotin molecule and reacting with a surface to which avidin has beenattached, or alternatively, and preferably, by covalent attachment tothe surface. The latter may be accomplished by derivatizing caged-biotinand biotin analogues at their carboxylic acid terminus. Many biotinderivatives have been described previously. For example, the surface canbe provided with biotin anti-ligands, e.g., antibiotin antibodies, whichspecifically bind the carboxyl arm of biotin without interfering withthe avidin-binding ureido ring of biotin.

Still another method for immobilizing the caged binding members of thepresent invention involves chemical derivatization or activation of thebinding member prior to attachment to the surface or linker. Forexample, when the surface is a polymer containing primary amines andbiotin is selected as the binding member, the N-hydroxysuccinimide esterderivative of biotin can react with the surface to give a biotin-surfacecomplex (U.S. Pat. No. 4,282,287).

Alternatively, and preferably, photoactivatable biotin and biotin analogderivatives will be covalently attached to the surface. To effect thistransformation, the biotin and biotin analogs may be derivatized attheir carboxylic acid terminus. Many biotin derivatives have beendescribed previously involving derivatization at the free carboxyl endof biotin. See, e.g., Bayer et al., Methods of Biochemical Analysis,vol. 26 (D. Glick, ed.), 1-45 (1980), which is incorporated herein byreference. For example, photoactivatable biotin derivatives may bereacted, in the presence of an activating reagent, such as acarbodiimine or BOP, with the amine groups of crosslinking groupspreviously immobilized on the surface to give the biotin attached to thesurface via an amide linkage. The active ureido ring of biotin, eitherfree or protected, is located far enough away from the site ofattachment that, when unprotected, binding with avidin is notsignificantly diminished.

It should be appreciated that the above discussion of exemplary surfaceattachment reactions is only illustrative of the general method forattaching caged binding members to a surface and should not be regardedin any way as limiting the applicability of the method to biotin, biotinanalogs, or specific crosslinking groups. Other types of binding membersalso amenable to the above attachment techniques include enzymes,antibodies, oligonucleotides and the like and are readily apparent toone skilled in the art.

IV. Properties of Binding Members and Caging Groups

The present method permits use of a wide variety of caged bindingmembers to effect the immobilization of anti-ligands on the surface. Themethod is generally applicable to such classes of compounds as enzymes,substrates, cofactors, immunoglobulins, antibodies, haptens, antigens,oligonucleotides, oligosaccharides, lectins, proteins, glycoproteins,etc., being the binding member provided that the selected derivative ofsuch species is activatable upon exposure to a suitable energy source.Moreover, the binding member can possess a multiplicity of binding sitesfor an anti-ligand or specific binding substance.

The binding member selected will have a high binding affinity either foran anti-ligand or a specific binding substance. Preferably, a specificbinding substance will provide a link between the binding member and theanti-ligand. Usually, the interactions between a binding member and aspecific binding substance and an anti-ligand or anti-ligand conjugatewill be noncovalent in nature. When a specific binding substanceprovides a link between the binding member and the anti-ligand, thespecific binding substance will be connected to the anti-ligand eithercovalently or through noncovalent interactions.

The binding member on a surface must have a strong affinity for ananti-ligand or specific binding substance to prevent migration or lossof the anti-ligand during wash steps. The affinity between the bindingmember and an anti-ligand or a specific binding substance is bestrepresented by the off-rate of anti-ligand or specific binding substancefrom a binding member. However, off-rates often are not convenientlyknown or determined. Therefore, binding affinity may also be representedby the affinity constant (K_(a)) for equilibrium concentrations ofassociated and dissociated configurations, i.e., K_(a)=[B-SBS]/[B][SBS], where [B], [SBS] and [B-SBS] are the concentrationsof the binding member (B), the concentration of specific bindingsubstance (SBS), and the concentration of associated complex (B-SBS),respectively, at equilibrium. An analogous definition of K_(a) applieswhen SBS is replaced with an anti-ligand (AL) or a specific bindingsubstance--anti-ligand conjugate (SBS-AL), etc. The affinity constantsof some sample classes of compounds suitable for use in the presentinvention are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        Affinities of Sample Binding Members and Specific Binding                     Substances (SBS).                                                             Binding Member   SBS       Affinity (K.sub.a M.sup.-1)                        ______________________________________                                        Membrane sites   Lectins   10.sup.6-7                                         Haptens          Antibodies                                                                              10.sup.5-11                                        Antigenic        Antibodies                                                                              10.sup.5-11                                        determinants                                                                  Biotin           Avidin    10.sup.15                                          Iminobiotin      Avidin    10.sup.11                                          2-thiobiotin     Avidin    10.sup.13                                          Dethiobiotin     Avidin    10.sup.13                                          1'-N-methoxy-carbonylbiotin                                                                    Avidin    10.sup.7                                           methyl ester                                                                  3'-N-methoxy-carbonylbiotin                                                                    Avidin    10.sup.9                                           methyl ester                                                                  ______________________________________                                         *References: U.S. Pat. No. 4,282,287; Green, "Avidin" in Advances in          Protein Chemistry, Academic Press, vol. 29, 105 (1975).                  

Preferably, the affinity constant between the activated binding memberand another species, i.e., a specific binding species, an anti-ligand,or anti-ligand conjugate, will be greater than about 10⁷ M⁻¹. Morepreferably, the K_(a) will be greater than about 10¹¹ M⁻¹, and mostpreferably, the K_(a) will be about 10¹⁵ M⁻¹ or greater. Likewise, whena specific binding substance is used, the affinity constant between thespecific binding substance and an anti-ligand or anti-ligand conjugatewill have substantially the same ranges as given above.

An activated (uncaged) binding member is considered to have a relativelystrong (high) binding affinity for another species, i.e., a specificbinding substance, an anti-ligand, or a conjugate of an anti-ligand,when the K_(a) between the binding member and the other species is atleast about three orders of magnitude greater than the correspondingK_(a) between the caged binding member and the other species. Similarly,a caged binding member is considered to have a relatively low bindingaffinity for another species, i.e., specific binding substance,anti-ligand or anti-ligand conjugate, when the K_(a) between the cagedbinding member and the other species is about three orders of magnitudeless than the corresponding K_(a) for the activated binding member.Preferably, the affinity constant for the caged binding member will beat least five orders of magnitude lower than the corresponding activatedbinding member's affinity constant. Most preferably, the bindingconstant for the caged binding member will be even lower, e.g., sevenorders of magnitude lower, than the corresponding activated bindingmember's affinity constant. However, the suitability of a given cagedbinding member/binding member pair for practice of the invention isdetermined ultimately by whether the selected pair permits properoperation of the invention.

A preferred embodiment of the present invention employs biotin andbiotin analogs as the binding members. Typical biotin analogs includedethiobiotin, iminobiotin, 2-thiobiotin, azabiotin, biocytin, and biotinsulfone, and other compounds readily apparent to one skilled in the art.Exemplary biotin analogs include, but are not limited by, thosepresented in Table 2. Other biotin analogs are presented in N. Green,"Avidin" in Advances in Protein Chemistry, Vol 29, Acad. Press, p.85-133 (1975), which is incorporated by reference herein. Biotin analogsinclude compounds and structures in which biotin is bound to anotherspecies, such as a surface, as long as the analog has a binding affinityfor avidin that is similar to that of biotin. The biotin or biotinanalogs may be subsequently reacted with avidin compounds, streptavidin,and analogues thereof.

Typical examples of avidin and avidin analogs include, but are notlimited to, the avidin found in eggs and streptavidin. Streptavidin is atypical example of an avidin analog and is a bacterial biotin-bindingprotein which has physical characteristics similar to those of eggavidin, despite considerable differences in composition.

                  TABLE 2                                                         ______________________________________                                        Biotin and Biotin Analogs                                                                             Name                                                  ______________________________________                                         ##STR2##               Biotin                                                 ##STR3##               Iminobiotin                                            ##STR4##               Dethiobiotin                                           ##STR5##               2'-thiobiotin                                          ##STR6##               Azabiotin                                              ##STR7##               Bisnorazabiotin                                       ______________________________________                                         Reference: N. Green, "Avidin," in Advances in Protein Chemistry, Vol. 29,     Academic Press, p. 85-133 (1975).                                        

Binding members other than biotin and their corresponding specificbinding substances may be employed in the present invention. By way ofexample and not limitation, some alternative embodiments of theinvention follow:

1. Caged Cyclic AMP/ Anti-cAMP Antibodies

High-affinity polyclonal antibodies to cAMP are produced by immunizingwith 2'O-monosuccinyl adenosine 3', 5' cyclic monophosphate conjugatedto a protein such as bovine serum albumin or thyroglobulin. Purifiedpolyclonal antibodies are prepared by affinity chromatography usingagarose gel to which 2'O-monosuccinyl adenosine 3',5' cyclicmonophosphate has been conjugated. The K_(a) of polyclonal antibodies tocAMP is in the range of 10¹⁰ to 10¹² M⁻¹.

A photoactivatable analog of cAMP has been previously described (Nerboneet al., Nature (1984) 310:74). It is unlikely that polyclonal antibodiesagainst cAMP have high affinity for the photoactivatable analog of cAMP.If the polyclonal antibodies should cross-react with thephotoactivatable analog of cAMP, monoclonal antibodies can be producedwhich discriminate between cAMP and the photoactivatable cAMP analog.

The 2'O-monosuccinyl derivative of the photo-activatable cAMP analog isattached to a surface through the free carboxyl of the succinyl group asdescribed above. Specific regions of the surface are illuminatedresulting in the removal of the protecting group from the cAMP.Anti-ligands which have been conjugated to anti-cAMP antibodies arereacted with the surface. The anti-ligands are immobilized only at thepredefined regions of the surface that were illuminated.

2. Caged Tetrabhydrofolate/ Folate Binding Proteins

N₅ -(Nitroveratryloxycarbonyl)tetrahydrofolate is activated at itsglutamyl gamma-carboxylate with a carbodiimide reagent and coupled to anamino-derivatized surface. Desired predefined regions on the surface areirradiated with light suitable for deprotection of the NVOC group. Inthe illuminated regions, the NVOC group is removed to producetetrahydrofolate bound to the surface. High-affinity folate bindingproteins derived from human erythrocyte membranes (Antony et al., J.Clin. Invest. (1987) 80:711-723; K_(a) =3×10¹¹ M⁻¹ fortetrahydrofolate), crosslinked to a desired anti-ligand, are thenimmobilized on the selected regions of the surface.

3. Caged Mannose/ Concanavalin A

8'-(Trichlorosilyl)octyl 6-(nitroveratryloxy)-a-D-mannoside iscovalently attached to a silica or glass surface by methods well-knownto those skilled in the art. Predefined regions of the surface areirradiated with light suitable for deprotection of the nitroveratryloxygroup. In the irradiated regions, the protecting group is removed toproduce octyl-e-D-mannoside bound to the surface. In unexposed areas,the 6-(nitroveratryloxy) group protects the mannoside from binding tothe lectin. Concanavalin A, conjugated to a desired anti-ligand, isadded to the surface and binds to those mannose units on the surfacethat have been deprotected. The anti-ligand is thereby immobilized onthe desired regions of the surface.

The above modes for practicing the invention are examples only. Thoseskilled in the art will recognize that any pair of (i) binding memberand specific binding substance, (ii) binding member and anti-ligand oranti-ligand conjugate, or (iii) specific binding substance andanti-ligand or anti-ligand conjugate may be used. The only restrictionson the choice of binding member, specific binding substance andanti-ligand or anti-ligand conjugate are that: (1) the binding memberhas a high affinity for the specific binding substance, anti-ligand oranti-ligand conjugate selected, (2) the binding member can be "caged"with a removable protecting group, and (3) the caged binding member hasa relatively low affinity for specific binding substances, anti-ligands,or anti-ligand conjugates, and any species which interfere with practiceof the invention.

A. Caging Groups

Many different protecting (caging) groups can be employed for modifyingbinding members to give the caged binding members of the presentinvention. The protecting groups should be sterically large enough toreduce the affinity of the binding member for anti-ligands or specificbinding substances to permit operability of the invention, althoughprotecting groups utilizing other types of interactions, such aselectronic (i.e., Van der Waals), hydrophobic, etc., could be used. Theselection of suitable caging groups will depend upon the size andchemical nature of the binding member chosen and will be readilyapparent to one skilled in the art.

In a preferred embodiment, the caging groups will be photoactivatable.The properties and uses of photoreactive caged compounds have beenreviewed. See, J. McCray, et al., Annu. Rev. Biophys., Biophys. Chem.,18: 239-70 (1989), which is incorporated herein by reference.Preferably, the photosensitive cages will be activatable by low energyultraviolet or visible light. In some embodiments, however, activationmay be performed by the methods discussed later, including localizedheating, electron beam techniques, laser pumping, and oxidation orreduction with microelectrodes, Alternatively, the reactive group may beactivatable by electron beam lithography, X-ray lithography, or anyother radiation. Suitable reactive groups for electron beam lithographyinclude sulfonyl compounds. Other methods may be used including, forexample, exposure to an electric current source, preferably usingmicroelectrodes directed to the predefined regions of the surface whichare desired for activation. Other reactive groups and methods ofactivation may be used in light of this disclosure.

A further preferred embodiment of the present invention employsphotoactivatable N-derivatives of biotin and biotin analogs to reducethe natural affinity of biotin for other compounds, such as avidin usedas a specific binding substance, until the groups attached to theN-positions are photoremoved. A few references describe N-derivatizationof biotin and biotin analogs. See, Kohn et al., J. Org. Chem. (1977)42:941-948, and Knappe et al., Biochemische Zeitschrift, 35, 168-176(1961). However, none of these references provide photoactivatablebiotin derivatives. The use of a photosensitive biotin derivative,photobiotin, has been previously described for labelling proteins andnucleic acids. Lacey, E. Anal. Biochem,, 163: 151-8 (1987); Forster, A.C. Nucleic Acids Res., 13: 745-61 (1985). However, photobiotin is aderivative of the carboxylate terminus of biotin, which is located awayfrom the recognition site and, hence, does not significantly reducebinding to avidin or streptavidin.

Many, although not all, of the photosensitive protecting groups will bearomatic compounds. Suitable photoremovable protecting groups aredescribed in, for example, McCray, et al., Patchornik, J. Am. Chem. Soc.(1970) 92:6333 and Amit, et al., J. Org. Chem. (1974) 39:192, which areincorporated herein by reference. See, also, Calbiochem Catalog, (SanDiego, Calif., 1989), p. 244-247. More preferably, the photosensitivegroup will be a nitro benzylic compound, such as o-nitrobenzyl orbenzylsulfonyl groups. In a preferred embodiment,6-nitroveratryloxycarbonyl (NVOC) and its derivatives, such as6-nitropiperonyloxycarbonyl (NPOC),α,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ) or 1-pyrenylmethyl may beemployed.

When the selected binding member is biotin or a biotin analog,photosensitive protecting groups may be provided at the N-1', N-3' or toan oxygen, imino, or sulfur group at the 2'-C position of the ureidoring. When the protecting group is attached to a 2'-C-O- position, thegroup will preferably be an o-nitro benzylic group having a hydrogenatom at the alpha benzylic position. In such case, the biotin or biotinresidue is an imidazolidine group. When the protecting group is attachedto a 1'-N or 3'-N atom, the protecting group will preferably be ano-nitro benzylic group having a hydrogen atom bound to the alpha carbonatom and optionally an oxycarbonyl group linking the alpha carbon atomthrough the oxygen atom. In the latter case, the derivatized nitrogenatom of the imidazolidone group will be bound to the carbon atom of theoxycarbonyl group.

A preferred embodiment of the invention has the following formula:##STR8## where X and Z are hydrogen, or oxycarbonyls of lower alkyl,aryl, or benzyl groups, provided that X and Z are not both hydrogen; Ris hydrogen, lower alkyl, aryl, carboxylate, alkyl formate, arylformate, formamide, N-alkylformamide, N-succinimidyl, hydroxyl, alkoxyl,thiol, thioether, disulfide, hydrazide or an amine group; U is O, S, orNH; Y is sulfur, oxygen, methylene, carbonyl, or a sulfinyl, or sulfonylgroup, or Y represents two hydrogen atoms attached to the respectivecarbons; and n=0-7. Also, inorganic and organic acid addition salts ofthe above compounds can be employed. Furthermore, R can represent asurface or a surface provided with a suitable crosslinking group. Inanother preferred embodiment, R is hydrogen, lower alkyl, aryl,carboxylate, alkyl formate, aryl formate, formamide, N-alkylformamide,N-succinimidyl, hydroxyl, alkoxyl, thiol, thioether, disulfide,hydrazide or an amine group connected to a linking group of a suitablelength, such as monomer, dimer, trimer, or oligomer of 6-aminocaproicacid, an oligomer of ethylene glycol having up to 10 unitsdioxadodecane-propyl, or other suitable linkers. A more preferredembodiment is when R is methyl formate or p-nitrophenyl formate. Afurther preferred embodiment is when U is O, Y is S, and n=4. Apreferred embodiment is when Y represents two hydrogen atoms attached torespective carbons, which eliminates the lower ring leaving a methylgroup and a hexanoic acid group: ##STR9##

A further preferred embodiment is when X or Z is a nitro aromaticcompound containing a benzylic hydrogen ortho to the nitro group. Astill further preferred embodiment is when X or Z has the formula:##STR10## where R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl,halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl,formate, sulfonate, formamido or phosphido groups. A further preferredembodiment is when X or Z is a nitroveratryloxycarbonyl group.

Most preferred embodiments are when X is 6-nitroveratryloxycarbonyl, Zis hydrogen, and R is methyl formate or p-nitrophenyl formate.

Another preferred embodiment is when X or Z is a ring-disubstitutedbenzyloxycarbonyl group having the formula: ##STR11## where R₁ and R₂are hydrogen, lower alkyl, aryl, benzyl, pyrenyl, halogen, hydroxyl,alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido orphosphido groups, and R₃ and R₄ are hydrogen, lower alkyl, aryl, benzyl,halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl,formate, formamido or phosphido groups. More preferably R₁ and R₂ aremethoxy groups. More preferably R₃ and R₄ are methyl groups. A mostpreferred embodiment is when R₁ and R₂ are methoxy groups and R₃ and R₄are methyl groups.

A further preferred embodiment is when X has the formula: ##STR12##where R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, halogen,hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate,formamido or phosphido groups. A still further preferred embodiment iswhen X is a nitroveratryl group.

Most preferred embodiments are when X is 6-nitroveratryl, U or W ishydrogen, and R is methyl formate or p-nitrophenyl formate.

A further preferred embodiment is when X is a ring-disubstituted benzylgroup. A more preferred embodiment is when the ring-disubstituted benzylgroup has the formula: ##STR13## where R₁, R₂, R₃, and R₄ are hydrogen,lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether,amino, nitro, carboxyl, formate, formamido or phosphido groups. Mostpreferred embodiments are when R1 and R₂ are methoxy and R₃ and R₄ aremethyl.

Another preferred embodiment is when the composition has the followingformula: ##STR14## where X is hydrogen, lower alkyl, aryl, or benzyl; Uand W are hydrogen, lower alkyl, aryl, or benzyl groups, provided thatonly one of U and W is present; R is hydrogen, lower alkyl, aryl,carboxylate, alkyl formate, aryl formate, formamide, N-alkyl formamide,N-succinimidyl, hydroxyl, alkoxyl, thiol, thioether, disulfide,hydrazide, or amine groups; Y is sulfur, oxygen, methylene, carbonyl, ora sulfinyl, or sulfonyl group, or Y represents two hydrogen atomsattached to the respective carbons; and n=0-7. Also, inorganic andorganic acid addition salts of the above compounds are suitable. A morepreferred embodiment is when Y is sulfur and n=4.

Clearly, many photosensitive protecting groups are suitable for use inthe present inventive methods. Some examples of acceptablephotosensitive protecting groups are presented in Table 3. Also, someexample protecting groups and their corresponding wavelengths fordeprotection are provided in Table 4.

                  TABLE 3                                                         ______________________________________                                        Example Protecting Groups                                                                          Name                                                     ______________________________________                                         ##STR15##                                                                                          ##STR16##                                                ##STR17##                                                                                          ##STR18##                                                ##STR19##                                                                                          ##STR20##                                                ##STR21##                                                                                          ##STR22##                                                ##STR23##                                                                                          ##STR24##                                                ##STR25##                                                                                          ##STR26##                                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Example protecting groups and their deprotection wavelengths                                         Deprotection                                           Group                  Wavelength                                             ______________________________________                                        Nitroveratryloxycarbonyl                                                                             UV (300-350 nm)                                        Nitrobenzyloxycarbonyl UV (300-350 nm)                                        Dimethyldimethoxybenzyloxycarbonyl                                                                   UV (280-300 nm)                                        5-Bromo-7-nitroindolinyl                                                                             UV (420 nm)                                            o-Hydroxy-α-methyl cinnamoyl                                                                   UV (300-350 nm)                                        2-Oxymethylene anthraquinone                                                                         UV (350 nm)                                            ______________________________________                                    

B. Irradiation

Once the surface is covered with a plurality of caged binding members,selected regions of the surface may be irradiated to provide activatedbinding members. Predefined regions of the surface may be selectivelyactivated by electron beam lithography, ion beam lithography, X-raylithography, or any other radiation method. In a preferred embodiment,the radiation is UV, near IR, or visible light. The light source may becoherent or noncoherent. The protective group may alternatively be anelectrochemically-sensitive group which may be removed in the presenceof an electric current.

In some embodiments, the exposed area is less than about 1 cm² or lessthan about 1 mm². In preferred embodiments the exposed area is less thanabout 10,000 μm² or, more preferably, less than about 100 μm². Spacesbetween activated regions are not critical and will generally be greaterthan about 1 μm.

When photoactivatable binding members are used, they are preferablyexposed to light through a suitable mask using photolithographictechniques well known in the semiconductor industry and described in,for example, Sze, VLSI Technology, McGraw-Hill (1983), which isincorporated herein by reference. In one embodiment, the mask is atransparent support material coated with a layer of opaque material.Portions of the opaque material are removed, leaving opaque material inthe precise pattern desired on the substrate surface. The mask isbrought into close proximity with or directly into contact with thesurface. Openings in the mask correspond to locations on the surfacewhere it is desired to photoremove protecting groups from the bindingmembers. Alignment may be performed using conventional alignmenttechniques in which alignment marks are used to accurately overlaysuccessive masks with previous patterning steps. Other alignmenttechniques may be used, for example, interferometric techniques such asthe one described in Flanders, et al., "A New Interferometric AlignmentTechnique," App. Phys. Lett. (1977) 31:426-428, which is incorporatedherein by reference.

To enhance contrast of light applied to the substrate it may bedesirable to provide contrast enhancement materials between the mask andthe substrate. This contrast enhancement layer may comprise a moleculewhich is decomposed by light such as quinone diazide.

The light may be from a conventional incandescent source, an arc lamp, alaser, or the like. If noncoherent sources of light are used it may bedesirable to provide a thick- or multi-layered mask to prevent spreadingof the light on the substrate. Generally, lasers may be preferablebecause they can more easily provide wavelengths particularly suited fora chromophore of the photosensitive group.

While the invention is illustrated primarily herein by way of the use ofa mask to illuminate the substrate, other techniques may also be used.For example, the substrate may be rotated under a modulated laser ordiode light source. Such techniques are discussed in, for example, U.S.Pat. No. 4,719,615, which is incorporated herein by reference.

The substrate may be irradiated either in contact with or not in contactwith a solution and, preferably, is irradiated in contact with thesolution. The solution may contain reagents to prevent by-products ofirradiation from interfering with subsequent binding reactions. Suchby-products might include, for example, carbon dioxide, nitrosocarbonylcompounds, styrene derivatives, indole derivatives, and products oftheir photochemical reactions. Reagents added to the solution mayinclude, for example, acidic or basic buffers, thiols, substitutedhydrazines and hydroxylamines, reducing agents (e.g., NADH or bisulfiteion) or reagents known to react with a given functional group (e.g.,aryl nitroso+glyoxylic acid→aryl formhydroxamate+CO₂). Preferably,however, protecting groups will be selected which do not causesignificant interferences with the binding reactions. Also, wash stepswill be incorporated so that the by-products do not interfere with thereactions.

In a preferred embodiment, a surface provided with a plurality of sitesoccupied by photosensitive N-derivatives of biotin or biotin analogs isexposed to a desired light pattern to cause loss of some or all of thephotosensitive protecting groups at predefined regions on the surface.Such irradiation of the N-derivatized biotin compounds of the presentinvention leads to formation of surface-bound biotin or biotin analogshaving a strong specific binding affinity for avidin or avidin analogs.The specific binding affinity of biotin and avidin is one of thestrongest known between macromolecules (K_(a) =10¹⁵ M⁻¹). This bindingpersists when the carboxyl terminus of biotin is attached to anotherentity, e.g., a surface, or when avidin is attached to another molecule.Avidin possesses four subunits having specific binding affinity forbiotin molecules. For example, deprotected biotin sites may be incubatedwith avidin or an avidin conjugate of an anti-ligand, e.g., an antibody,to provide a localized concentration of the desired anti-ligand on thesurface. When incubation with avidin alone is performed, it is necessaryto further incubate the resulting product with a preselected specieshaving specific binding affinity for avidin, e.g., a biotinylatedanti-ligand. Thus, biotinylated anti-ligands can be bound to the freesites of avidin to afford anti-ligands immobilized at predefined regionson the surface. For a general discussion of the use of the biotin-avidininteraction in molecular biology, see Bayer, et al. Once localization ofthe anti-ligand is complete, the light pattern can be changed and thesame or a different anti-ligand can be localized at other discrete siteson the surface.

V. Attachment of Anti-ligands

An anti-ligand is one or more molecules that recognize a particularligand in solution. Examples of ligands that can be investigated by thisinvention include, but are not restricted to agonists and antagonistsfor cell membrane receptors, toxins and venoms, viral epitopes,antigenic determinants, hormones, hormone receptors, steroids, peptides,enzymes, substrates, cofactors, drugs, lectins, sugars,oligonucleotides, oligosaccharides, proteins, and monoclonal andpolyclonal antibodies.

Anti-ligands that mediate a biological function on binding withparticular ligand(s) are of most interest. Suitable anti-ligands includerelatively small, single molecules, such as cofactors, which showspecific binding properties. Typically, anti-ligands will be greaterthan about 100 daltons in size and more typically will be greater thanabout 1kD in size. Other examples of anti-ligands include, but are notrestricted to, the common class of receptors associated with the surfacemembrane of cells and include, for instance, the immunologicallyimportant receptors of B-cells, T-cells, macrophages and the like. Otherexamples of anti-ligands that can be investigated by this inventioninclude but are not restricted to hormone receptors, hormones, drugs,cellular receptors, membrane transport proteins, steroids, peptides,enzymes, substrates, cofactors, vitamins, lectins, sugars,oligonucleotides, oligosaccharides, viral epitopes, antigenicdeterminants, glycoproteins, and immunoglobulins, e.g., monoclonal andpolyclonal antibodies.

In a preferred embodiment, the anti-ligand will be a biotinylatedreceptor which binds specifically to avidin. Many biotinylatedanti-ligands and biotinylating reagents are commercially available.(See, for example, Vector Laboratories, Inc., Catalog, Burlingame,Calif.) Methods for biotinylating desired anti-ligands are well-known inthe art and are described, for example, in Bayer, et al.

In a preferred embodiment a plurality of anti-ligands is immobilized ona surface by first attaching photoreactive caged binding members to thesurface. The caged binding members on a predefined region of the surfaceare exposed to light to give binding members having a high affinity fora specific binding substance. The activated binding members on thepredefined region are then incubated with the specific bindingsubstance, the surface is washed free of unbound specific bindingsubstance, and the surface is incubated with a desired anti-ligand oranti-ligand conjugate. The exact incubation conditions, e.g., time,temperature, pH, will depend upon the species used and will be readilyapparent to one skilled in the art. After washing the surface free ofunbound anti-ligand, the above steps can be repeated on a differentregion of the surface.

In another embodiment of the invention a plurality of anti-ligands isimmobilized on a surface as described above, except the attachment ofanti-ligands to specific binding substance is carried out prior tointroducing the specific binding substance to the surface.

In a further embodiment the anti-ligand is a monoclonal or polyclonalantibody. In a still further preferred embodiment the anti-ligand is abiotinylated antibody or biotinylated receptor.

A most preferred embodiment of the invention is when the binding memberis biotin or a biotin analog and the specific binding substance isavidin or an avidin analog.

VI. Screening and Assays

A surface prepared according to the methods described above can be usedto screen for ligands having high affinity for immobilized anti-ligands.Screening can be performed by immobilizing a plurality of anti-ligandson predefined regions of a surface by the methods described above. Asolution containing a marked (labelled) ligand is introduced to thesurface and incubated for a suitable period of time. The surface is thenwashed free of unbound ligand and the anti-ligands having high affinityfor the ligand are identified by identifying those regions on thesurface where markers are located. Suitable markers include, but are notlimited to, radiolabels, chromophores, fluorophores, chemiluminescentmoieties, and transition metals. Alternatively, the presence of ligandsmay be detected using a variety of other techniques, such as an assaywith a labelled enzyme, antibody, and the like. Other techniques usingvarious marker systems for detecting bound ligand will be readilyapparent to those skilled in the art.

In a preferred embodiment, a substrate prepared as discussed above canbe exposed to a solution containing a marked ligand such as a markedantigen. The ligand can be marked in any of a variety of ways, but inone embodiment marking is effected with a radioactive label. The markedantigen binds with high affinity to an immobilized antibody previouslylocalized on the surface. After washing the surface free of unboundligand, the surface is placed proximate to x-ray film to identify theantibodies that recognize the antigen. Alternatively, a fluorescentmarker may be provided and detection may be by way of a charge-coupleddevice (CCD), fluorescence microscopy or laser scanning.

When autoradiography is the detection method used, the marker is aradioactive label, such as ³² P. The marker on the surface is exposed toX-ray film, which is developed and read out on a scanner. An exposuretime of about 1 hour is typical in one embodiment. Fluorescencedetection using a fluorophore label, such as fluorescein, attached tothe ligand will usually require shorter exposure times.

Quantitative assays for ligand concentrations can also be performedaccording to the present invention. In a direct assay method, thesurface containing localized anti-ligands prepared as described above,is incubated with a solution containing a marked ligand for a suitableperiod of time. The surface is then washed free of unbound ligand. Theamount of marker present at predefined regions of the surface is thenmeasured and can be related to the amount of ligand in solution. Methodsand conditions for performing such assays are well-known and arepresented at, for example, L. Hood, et al., Immunology,Benjamin/Cummings (1978) and E. Harlow, et al., Antibodies. A LaboratoryManual, Cold Spring Harbor Laboratory, (1988). See, also U.S. Pat. No.4,376,110 for methods of performing sandwich assays. The preciseconditions for performing these steps will be apparent to one skilled inthe art.

A competitive assay method can also be employed by the presentinvention. Such a method involves immobilizing anti-ligands onpredefined regions of a surface as described above. An unmarked ligandis then bound to anti-ligands on the surface having specific bindingaffinity for the ligand. A solution containing marked ligand is thenintroduced to the surface and incubated for a suitable time. The surfaceis then washed free of unbound reagents and the amount of markerremaining on the surface is measured. Alternatively, marked and unmarkedligand can be exposed to the surface simultaneously. The amount ofmarker remaining on predefined regions of the surface can be related tothe amount of unknown ligand in solution.

Use of the invention herein is illustrated primarily with reference toscreenings of ligands for anti-ligands and assays for ligands. Theinvention will, however, find many other uses. For example, theinvention may be used in information storage (e.g., on optical disks),production of molecular electronic devices, production of stationaryphases in separation sciences, and in immobilization of cells, proteins,lectins, nucleic acids, polysaccharides and the like in any desiredpattern on a surface via molecular recognition of a specificanti-ligand.

The invention has been described primarily with reference to the use ofphotoremovable protecting groups, but it will be readily recognized bythose of skill in the art that other types of groups can be used andthat other sources of radiation can also be used. For example, in someembodiments it may be desirable to use protecting groups sensitive toelectron beam irradiation, X-ray irradiation, X-ray lithography, orcombinations thereof. Alternatively, the group could be removed byexposure to an electric current.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reviewing the above description. Thescope of the invention should, therefore, be determined not withreference to the above description, but should instead be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

VII. Examples

The following examples of preferred embodiments of the present inventionare presented by way of illustration only and do not suggest that theabove- described methods and compositions are in any way limited by thespecific examples set forth below.

Synthesis of Photoreactive N-1'-Derivatives of Biotin

Methods for the preparation of acyl imidazolidones, such as biotinderivatives, are well-known. See, for example, Kohn, et al., J. Org.Chem. (1977), 42, 941-948 and Knappe, et al., Biochem. Z. (1961), 335,168-176. Treatment of biotin methyl ester with methyl chloroformate inrefluxing chloroform (no base) for 72-80 h afforded a mixture heavilyfavoring the N-1'-derivative. Under similar conditions, the use ofnitroveratryloxycarbonyl (NVOC) chloride (Amit, et al., J. Org. Chem.(1974), 39, 192-196) gave N-1'-(nitroveratryloxycarbonyl)-biotin methylester (NVOC-biotin-OMe) in 47% yield after chromatography andcrystallization. Likewise, N-1'-(nitroveratryloxycarbonyl)-biotinp-nitrophenyl ester (NVOC-biotin-ONP) was obtained in 39% yield.

The structural assignment is based on precedent as well as spectroscopicproperties. See, for example, E. Becker, High Resolution NMR, 2nd ed.,Acad. Press (1980). In particular, the 1H NMR spectrum readilydifferentiates the ring fusion protons bearing 1) a urea nitrogen (ca4.2 ppm) and 2) an imide nitrogen (ca 4.8 ppm). Through the use of COSY(Derome, Modern NMR Techniques for Chemistry Research, Pergamon Press,Oxford (1987)) on N-1'-(nitroveratryloxycarbonyl)-biotin p-nitrophenylester, it was determined that the former ring fusion proton is vicinalto a methine adjacent to sulfur, and that the latter is vicinal to amethylene adjacent to sulfur.

Using the conditions reported by Kohn, et al. biotin methyl ester issimilarly derivatized with dimethyldimethoxycarbobenzyloxycarbonyl (DDZ)chloride or 1-pyrenylmethyloxycarbonyl (PYROC) chloride to afford thephotolabile compounds N-1'-(dimethyldimethoxycarbobenzyloxycarbonyl)biotin methyl ester (DDZ-biotin-OMe) andN-1'-(1-pyrenylmethyloxycarbonyl) biotin methyl ester(PYROL-biotin-OMe), respectively. The methods of Knappe, et al., andKohn, et al., can be employed to prepare analogous compounds and areincorporated by reference herein.

Example A Preparation of NVOC-biotin-OMe(N-1'-(6-nitroveratryloxycarbonyl)-biotin methyl ester) ##STR27##

2.00 g (8.19 mmol) of D-biotin was added to a methanolic HCl solutionprepared from 2.5 ml of acetyl chloride in 40 ml of anhydrous methanol.After stirring for 15 hours, the solvent was removed under reducedpressure to afford 2.11 g of the product biotin-OMe (biotin methylester) as a white solid, MP 116°-118° C. (100% yield).

NVOC-biotin-OMe was prepared from biotin via the intermediate, biotinmethyl ester (biotin-OMe) by either of two methods.

1. A solution of 1.00 g (3.87 mmol) of biotin-OMe and 1.60 g (5.81 mmol)of 6-nitroveratryloxycarbonyl chloride in 10 ml of chloroform was heatedto reflux for 50 hours. The product was purified via flash-columnchromatography on silica gel (3% methanol, 3% acetone, 94% chloroform aseluent) to afford 0.90 g of NVOC-biotin-OMe as a yellow solid (47%yield, 84% yield based on unreacted starting biotin-OMe), MP 199°-203°C., and 0.44 g of recovered biotin-OMe (44% recovery). The product,NVOC-biotin-OMe, was recrystallized from methylene chloride/ether.

2. A solution of 3.4 g (12 mmol) of 6-nitroveratryloxycarbonyl chloridein 40 ml of methylene chloride was added to a solution of 1.1 ml (14mmol) of pyridine and 1.3 g (13 mmol) of phenol in 15 ml of methylenechloride cooled to 0° C. The reaction mixture was allowed to warm toroom temperature and was stirred for 19 hours. The solution waspartitioned between methylene chloride and 1N HCl, the organic phase wasseparated and dried with magnesium sulfate, and the solvent was removedunder reduced pressure to give 4.1 g of a brown oil. Purification viaflash-column chromatography on silica gel (90% methylene chloride/10%hexane as the eluent) afforded 2.0 g of the product, 6-nitroveratrylphenyl carbonate, as a colorless oil.

A mixture of 399 mg (1.20 mmol) of 6-nitroveratryl phenyl carbonate in 5ml of chloroform and 202 mg (0.782 mmol) of biotin-OMe were heated toreflux. After 50 hours TLC showed that no reaction occurred, therefore35 mg of 60% sodium hydride (0.88 mmol) was added in-two equal portionsover 15 minutes. After an additional 16 hours at reflux, the reactionwas quenched with 3 drops of glacial acetic acid. The product waspurified via flash-column chromatography on silica gel (3% methanol, 3%acetone, 94% chloroform as the eluent) to afford 264 mg of the product,NVOC-biotin-OMe, as an off-white solid (68% yield, 75% yield based onrecovered unreacted biotin-OMe) and 20 mg of recovered biotin-OMe (10%yield).

Example B Preparation of NVOC-biotin-ONP(N-1'-(6-nitroveratryloxycarbonyl)-biotin para-nitrophenyl ester)##STR28##

A mixture of 0.340 g (0.93 mmol) of biotin p-nitrophenyl ester(purchased from Sigma Chemical Co., St. Louis) and 0.450 g (1.63 mmol)of 6-nitroveratryloxycarbonyl chloride in 4 ml of chloroform was heatedto reflux for 65 hours. The mixture was purified via flash-columnchromatography on silica gel (3% methanol, 3% acetone, 94% chloroform asthe eluent) to produce 0.231 g of NVOC-biotin-ONP as a beige solid (39%yield, 93% yield based on unreacted biotin-ONP), MP 203°-205° C., and0.21 g of recovered unreacted biotin-ONP (58% yield). The product wasfurther purified via recrystallization from chloroform/hexane.

Example C Preparation of NPOC-biotin-ONP(N-1'-(6-nitropiperonyloxycarbonyl)-biotin para-nitrophenyl ester)##STR29##

A mixture of 0.365 g (1.00 mmol) of biotin p-nitrophenyl ester and 0.410g (1.58 mmol) of 6 -nitropiperonyloxycarbonyl chloride in 5 ml ofchloroform was heated at reflux for 62 hours. The product was purifiedvia flash-column chromatography on silica gel (3% methanol, 3% acetone,94% chloroform as the eluent) to produce 0.231 g of product as a beigesolid (39% yield, 93% based on unrecovered starting material), MP203°-205° C., and 0.21 g of recovered biotin-ONP (58% yield). Asdescribed, the product was further purified via recrystallization fromchloroform/hexane.

Example D Preparation of NVOC-DT-biotin-OMe(N-1'-(6-nitroveratryloxycarbonyl)-dethiobiotin methyl ester). ##STR30##

A slurry of 50 mg (0.19 mmol) of biotin-OMe and about 1 g of RaneyNickel active catalyst (50% solution in H₂ O from Aldrich Chemical Co.)in 4 ml of methanol was stirred at room temperature for one hour. Thereaction mixture was diluted with chloroform, filtered and the recoveredcatalyst was washed with methanol. The combined wash and filtrate werepartitioned between chloroform and saturated sodium chloride acidifiedto pH 2 with 1N HCl. The combined organic phases were dried overmagnesium sulfate, and the solvent removed under reduced pressure togive 34 mg of pure DT-biotin-OMe as a white solid, MP 68°-72° C. (77%yield).

A mixture of 34 mg (0.15 mmol) of DT-biotin methyl ester and 74 mg (0.27mmol) of 6-nitroveratryloxycarbonyl chloride in 3 ml of chloroform washeated to reflux for 15 hours. The products are purified viaflash-column chromatography on silica gel (3% methanol, 97% chloroformas the eluent) to afford 45 mg of a 1:3 mixture of products as a yellowsolid (65% yield), MP 152°-156° C.

Example E:

Preparation of NVOC-biotin-OH (N-1'-(6-nitroveratryloxycarbonyl)-biotin)##STR31##

A solution of 262 mg (0.527 mmol) of NVOC-biotin-OME, prepared in-byeither method of Example A, in 15 ml of tetrahydrofuran and 2 ml ofdimethylforamide was treated with 10 ml of 1 N HCl. The reaction mixturewas heated to reflux for 49 hours, cooled to room temperature, and thesolvent removed under reduced pressure. The crude product was purifiedvia flash-column chromatography on silica gel (10% methanol, 90%chloroform as the eluent) to afford 178 mg of the pure product,NVOC-biotin-OH, as a white solid (70% yield), MP 219°-223° C.

Example F Chromatographic Evidence for Photoremoval of the NVOC groupfrom NVOC-biotin-ONP

A 100 mM solution of NVOC-biotin-ONP in acetonitrile, or biotin inwater, was placed in a quartz cuvette with a 2.0 mm pathlength. Thecuvette was irradiated for two minutes at a power of 1 watt/cm² with a500 W Hg(Xe) arc lamp (Oriel #66142) having a 305 nm long pass filter(Oriel #51450). Illuminated and non-illuminated samples were thensubjected to reverse-phase HPLC.

Shown in FIGS. 1a-1d are chromatographs of illuminated biotin andilluminated NVOC-biotin-ONP. The results support the following: 1)biotin was unaffected by the illumination; and 2) NVOC-biotin-ONP wasconverted to Biotin-ONP by the illumination.

Example G:

Estimation of the Affinity of NVOC-biotin-OMe for Avidin Before andAfter Illumination

All procedures using NVOC-biotin-OMe were conducted in dim red light.NVOC-biotin-OMe was dissolved in dimethylformamide (DMF) to aconcentration 1 mM and diluted to 100 μM with phosphate-buffered salinepH 7.4. In order to remove any contaminants having high affinity foravidin, 2 ml of the NVOC-biotin-OMe solution was mixed with 1 ml ofpacked streptavidin-Sepharose-4B resin (SIGMA) which had been washedthoroughly to remove any non-covalently bound avidin. After stirring forthree hours, the resin was removed by centrifugation followed byfiltration (0.2 micron nylon filter). The concentration of theNVOC-biotin-OMe solution (measured by 350 nm absorbance) was notsignificantly reduced by the resin treatment.

Microtiter wells (Beckman EIA plates) were treated for 1 hr with 200 μlof 0.5 μg/ml of streptavidin in 10 mM sodium bicarbonate buffer (pH9.6). After removal of the streptavidin solution and washing withphosphate buffered saline (PBS)/0.05% Tween 20, the wells were incubatedfor 1 hr at room temperature with 200 μl of PBS containing variousconcentrations of illuminated (as detailed above) and non-illuminatedbiotin or streptavidin-Sepharose treated NVOC-biotin-OMe. The wells werethen washed with PBS/Tween 20 and incubated for 1 hr at room temperaturewith 200 μl of PBS containing ³ H-biotin (30 Ci/mmol, New EnglandNuclear). The wells were then washed with PBS/Tween and treated for 30minutes at room temperature with 200 μl of 10% trichloroacetic acid inwater. The radioactivity in 100 μl was then determined by liquidscintillation counting.

The results of a representative binding experiment are shown in FIG. 2.The experiment was done three times with similar results. The dataindicate that NVOC-biotin-OMe has very low affinity for avidin asindicated by the fact that pre-incubation of avidin with concentrationsof NVOC-biotin-OMe ("caged biotin") as high as 10⁻⁵ M had no significanteffect on the subsequent binding of ³ H-biotin. In addition, the resultsindicate that illumination of NVOC-biotin-OMe generates a biotinderivative ("IL caged biotin") that was nearly as effective as biotin inblocking the subsequent binding of ³ H-biotin. Combined with thechromatographic evidence above, the data indicate that illumination ofNVOC derivatives of biotin leads to removal of the NVOC group.

Example H Demonstration of Photoremoval of NVOC group from NVOC-BiotinAttached to a Membrane

Nitrocellulose membrane filters (Biorad) were reacted with 5% bovineserum albumin in Tris-buffered saline (TBS) for 3 hr at roomtemperature. The membranes were washed with TBS, cut into 1 cm² sectionsand then reacted for 3 hr at room temperature (in the dark) with 10%DMSO/100 mM sodium borate buffer (pH 8.6) alone, 10 mM ofbiotin-N-hydroxysuccinimidyl ester in 10% DMSO/100 mM sodium boratebuffer (pH 8.6), or NVOC-biotin-ONP in 50% DMSO/100 mM sodium boratebuffer (pH 8.6). After washing with TBS, half the sections that weretreated with NVOC-biotin-ONP were illuminated in a manner identical tothat described above. After washing with TBS, the membrane sections wereincubated for 1 hr at room temperature with TBS containing 0.1% bovineserum albumin and 0.1 μCi/ml ¹²⁵ I-streptavidin (Amersham). Afterwashing with TBS, radioactivity on the membrane section was quantitatedby gamma counting.

FIG. 3 shows the data for a representative experiment. Binding of ¹²⁵I-streptavidin was approximately 3-fold higher with biotinylatedmembranes than with control membranes. Binding of ¹²⁵ I-streptavidin tonon-illuminated NVOC-biotinylated membranes was not significantlydifferent from non-biotinylated control membranes. ¹²⁵ I-streptavidinbinding to illuminated NVOC-biotinylated membranes was approximatelyequal to that of biotinylated membranes. These data indicate thatmembrane-bound NVOC-biotin has low affinity for streptavidin and thatillumination greatly increases streptavidin binding by removing the NVOCgroup from the biotin group.

Immobilization of anti-ligands on solid supports

Example I Preparation of Caged-Biotin Glass Plates

Commercially available glass microscope slides were derivatized withN-BOC-aminopropyltriethoxy silane according to literature procedures(for example, see J. Chromatography, 1974, Vol. 97, p. 33). The slideswere incubated in a solution of 20% trifluoroacetic acid in methylenechloride for 30 minutes to remove the BOC protecting group. Afterwashing sequentially with methylene chloride, dimethylformamide, andethanol, the slides were neutralized by immersing in a solution of 10%diisopropylethyl amine in methylene chloride for 30 minutes and furtherwashed with methylene chloride.

N-BOC-6-aminocaproic acid was converted to the BOP-activated ester inpreparation for reaction with the derivatized glass slide. A solution ofa 197 mg (0.445 mmol) ofbenzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate(BOP) in 0.40 ml of dimethylformamide was added to a solution of 86 mg(0.37 mmol) of N-BOC-6-aminocaproic acid, 56 mg (0.4 mmol) of1-hydroxybenzotriazole hydrate (HOBT), and 0.14 ml (0.80 mmol) ofdiisopropylethyl amine in 0.40 ml of dimethylformamide. After 10 minutesthe resultant solution was diluted with 3.20 ml of dimethylformamide togive a 0.10M solution of the activated ester. The derivatized slideswere incubated with 0.5 ml of the activated ester solution and after twohours of coupling, were sequentially washed with dimethylformamide,ethanol and methylene chloride. The BOC protecting groups were removedfrom the aminocaproic acid moieties, and the slide was subsequentiallywashed as described above. Biotin derivatives were coupled to the glassplates by either of two methods, as illustrated for NVOC-biotin.

Method A (via the BOP ester)

The BOP derivative of NVOC-biotin-OH was prepared by adding a solutionof 40 mg (0.099 mmol) of BOP in 0.09 ml of dimethylsulfoxide to asolution of 43 mg (0.090 mmol) of NVOC-biotin-OH, 13 mg (0.096 mmol) ofHOBT, and 0.035 ml of diisopropylethyl amine in 0.090 ml ofdimethylsulfoxide. After five minutes, the resulting solid was dilutedto 1.80 ml with dimethylsulfoxide to give a 0.05M solution of theactivated ester. Approximately 0.5 ml of the activated ester solutionwas applied to the surface of the derivatized glass surface. After beingexposed to the activated ester for two hours, the slides were washedsequentially with dimethylformamide, ethanol, and methylene chloride toyield a NVOC-biotin-caproicpropyl derivatized surface.

Method B (via the ONP ester)

Approximately 0.5 ml of an 0.10 M solution of NVOC-biotin-ONP indimethylformamide was applied to the surface of the derivatized glassslide. After 2-24 hours, the slides were washed sequentially withdimethylformamide, ethanol, and methylene chloride to yield aNVOC-biotin-caproic-propyl derivatized surface.

Example J:

Photodeprotection of an NVOC-Biotin-Caproic-Propyl-derivatized slide,and subsequent labeling with a Fluorescein-Streptavidin conjugate

A glass microscope slide to which NVOC-biotin has been covalentlyattached via a caproic-propyl spacer, as described in the Example I, wasmounted on a custom flow cell and illuminated through a 500 μm×500 μmcheckerboard-pattern mask (Photo Sciences Inc., Torrance, Calif.) usingbroad-band UV/blue light. The light source was a 500 W Mercury arc lamp(Oriel Model 87330) equipped with a 350 nm-450 nm dichroic reflector andproduced actinic light having an intensity of 12 mW/cm² as measuredthrough a 360 nm bandpass filter. The derivatized surface was photolyzedin flowing dioxane for 15 minutes, removed from the flow cell, andsequentially rinsed in deionized water, ethanol, and methylene chloride.

After incubation for one hour in a solution containing filtered PBS, 1%BSA, 0.05% Tween 20, pH 7.4, the activated surface was treated with asolution containing a Fluorescein-derivative of Streptavidin (MolecularProbes; 10 μg/ml in PBS/BSA/Tween 20) for one hour at room temperature.The slide was vortexed twice in PBS, 0.05% Tween 20, pH 7.4 for 10minutes, rinsed with deionized water, and allowed to dry. The slide wasexamined with a scanning fluorescence microscope (Zeiss Axioskopequipped with a Newport Model PM500-C motion controller, aSpectra-Physics Model 2020 argon-ion laser producing a 488 nm excitationlight; and a 520 nm long-pass emission filter) interfaced with aphoton-counting device (Hamamatsu Model 9403-02 photomultiplier;Stanford Research Systems Model SR445 amplifier and Model SR430multichannel scaler, IBM compatible PC) to generate a two-dimensionalimage consisting of fluorescence intensity data as a function of x,yposition. An example of this technique is described in U.S. Pat. No.5,143,854, which is a continuation-in-part of now abandoned U.S. Ser.No. 362,901, filed Jun. 7, 1989, the patent and application areincorporated herein by reference. FIG. 4 shows an example of the imagesobtained.

The light squares indicate regions of high fluorescence intensityresulting from localization of the fluorescein label attached to theanti-ligand. This experiment demonstrates enhanced binding ofstreptavidin upon photodeprotection of NVOC-biotin coupled to a surfacewith a caproic-propyl spacer, and spatially-addressable immobilizationof an anti-ligand, such as streptavidin, by non-covalent means.

Example K Photodeprotection of an NVOC-Biotin-Caproic-Propyl-derivatizedslide, treatment with Streptavidin, and labeling with aFluorescein-Biotin conjugate

The microscope slide of Example I having NVOC-biotin covalently attachedvia a caproic-propyl spacer was illuminated and processed as in ExampleJ, except that after preincubation with the PBS/BSA/Tween 20 solution,the surface was treated with 10 μg/ml solution of Streptavidin (inPBS/BSA/Tween 20) for 40 minutes at room temperature, followed byincubation with a 1 μM solution of Fluorescein-Biotin(5-(N-((5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein, in PBS, pH 7.4) for 20minutes. The resulting slide was then washed, dried, and examined usinga scanning fluorescence microscope as described above. FIG. 5 shows anexample of the images obtained.

The light squares indicate regions of high fluorescence intensityresulting from localization of the fluorescein label attached to ligand,biotin. This experiment demonstrates the binding of a ligand (aFluorescein-Biotin complex) to streptavidin immobilized in aspatially-addressable manner.

Example L Photodeprotection of an NVOC-Biotin-Polyether-derivatizedslide and labeling with a Fluorescein-Streptavidin conjugate

A microscope slide to which 3-aminopropyltriethoxy silane had beenattached was treated with the BOP-activated ester of18-amino-6-aza-10,15-dioxa-5-ketooctadecanoic acid using a proceduresimilar to Example I. The resulting slide was then derivatized withNVOC-Biotin-ONP, and was illuminated, processed, and examined asdescribed in Example J. FIG. 6 shows an example of the images obtained.

This experiment demonstrates spatially-localized streptavidin bindingupon photodeprotection of NVOC-Biotin coupled to a surface using analternative linker, a polyether-glutaric-propyl moiety.

Example M Photodeprotection of an NPOC-Biotin-Caproic-Propyl-derivatizedslide and labeling with a Fluorescein-Streptavidin conjugate

The microscope slide to which NPOC-biotin-ONP had been covalentlyattached via a caproic-propyl spacer was illuminated, processed, andexamined as described in Example J. FIG. 7 shows an example of theimages obtained.

This experiment demonstrates spatially-localized streptavidin bindingupon photodeprotection of caged-biotin using a different protectinggroup, NPOC.

Example N:

Photodeprotection of an NVOC-Biotin-Caproic-Propyl-derivatized slide inaqueous buffer and labeling with a Bodipy-streptavidin conjugate

The microscope slide of Example I having NVOC-Biotin covalently attachedvia a caproic-propyl spacer was mounted on a custom flow cell andilluminated using the apparatus described in Example J. The derivatizedsurface was photolyzed in PBS, 1% BSA, 0.1% Tween 20 for 30 minutes at12 mW/cm², removed from the flow cell, and processed as described inExample K, except that Bodipy-streptavidin (Molecular Probes, 50 μg/ml)was used instead of the fluorescein conjugate. FIG. 8 shows an exampleof the images obtained.

This experiment demonstrates spatially-localized streptavidin bindingupon photodeprotection of NVOC-biotin in a different solvent, in thiscase an aqueous buffer.

Example O Evaluation of crosslinking groups of different lengths

A microscope slide derivatized with N-Boc-3-aminopropyltriethoxy silane,as in Example I, was functionalized with three different crosslinkers indifferent locations by selectively coupling 1, 2 or 3N-Boc-6-aminocaproic acid linkers to the surface via their BOP-activatedesters. This generated four distinct and well defined regions on thesurface of the slide having zero, one, two and three 6-aminocaproic acidlinkers (4, 11, 18 and 25 atom spacers, respectively) between thesurface and the terminal amino functionality used to bind to thecarboxyl group of the derivatized biotin. A 0.1 M solution of biotinp-nitrophenyl ester in dimethylformamide was subsequently coupled to theslide, and the relative binding affinity of streptavidin to thesurface-bound biotin was measured by incubating the slide withfluoresceinated streptavidin and measuring the fluorescence intensity asin Example J. The measured relative fluorescence was 38, 68, 85 and 100%(normalized to the fluorescence of area having three caproic linkers),respectively, for zero, one two and three caproic linkers, showing thata higher density of streptavidin was bound to an area of the slidederivatized with biotin spaced relatively far from the surface of theslide. FIG. 9 shows the fluorescence of fluoresceinated streptavidinbound to the glass slide derivatized in this experiment.

Example P Photodeprotection of an NVOC-Biotin-Caproic-Propyl-derivatizedslide and subsequent immobilization of antibodies on the derivatizedsurface

A microscope slide to which NVOC-Biotin had been covalently attached viaa caproic-propyl spacer was mounted on a custom flow cell andilluminated as described in Example J, except that a hand-cut maskconsisting of a horizontal stripe (approximately 2 mm wide) was used.After removal from the flow cell and rinsing, the slide was incubatedfor 30 minutes with PBS, 1% BSA, 0.05% Tween 20, pH 7.4, followed by 30minutes treatment with Streptavidin (10 μg/ml in PBS/BSA/Tween 20),rinsing with PBS, 0.05% Tween 20, 60 minutes incubation withbiotinylated Rabbit IgG (Vector Laboratories; 50 μg/ml in PBS/BSA/Tween20), and rinsing with PBS/Tween 20. The surface was then "capped" toprevent subsequent streptavidin binding to the multiple biotin moietieson the biotinylated IgG. That is, streptavidin was used to bind freebiotin on IgG. This was accomplished by re-treatment with Streptavidinsolution, rinsing with PBS/Tween 20, followed by incubation with freebiotin (Molecular Probes; 1 mM in PBS/Tween 20; 10 minutes), and a finalPBS/Tween 20 rinse. The slide was then remounted on the flow cell,photolyzed for 30 minutes in PBS/Tween 20 using a hand-cut maskconsisting of vertical stripes (approximately 2 mm wide), and processedas described above, except that biotinylated Mouse IgG (VectorLaboratories; 50 μg/ml in PBS/BSA/Tween 20; 30 minute incubation) wasused, and the "capping" steps were not repeated. The slide was thenrinsed with deionized water, allowed to dry, and beads of siliconegasket compound (Permatex Ultra Blue) were used to partition the slideinto three areas. After pre-incubation for 30 minutes with PBS/BSA/Tween20, the first area was treated with Fluorescein-labeled anti-Rabbit IgG(Vector Laboratories; made in goat; 100 μg/ml in PBS/BSA/Tween 20). Thesecond area was treated with Fluorescein-labeled anti-Mouse IgG (VectorLaboratories; made in horse; 100 μg/ml in PBS/BSA/Tween 20). The thirdarea was incubated with an equimolar mixture of the two secondaryantibodies. The slide was then vortexed twice in PBS/Tween 20 for twominutes, rinsed briefly with deionized water, and allowed to dry. Thedifferent regions of the slide were examined using the scanningfluorescence microscope described in Example J.

FIG. 10 shows examples of the images obtained from the glass slidederivatized in this experiment. FIG. 10a shows the region of the glassslide treated with fluorescein-anti-Rabbit IgG. As expected, thehorizontal stripe, which corresponds to the area where biotinylatedRabbit IgG bound, is intensely fluorescent indicating a high density ofbound fluorescein-anti-Rabbit IgG. The vertical stripes in this regionare faintly visible, which may be due to the slight cross reactivity ofthe secondary antibodies. FIG. 10b shows the region of the glass treatedwith Fluorescein-anti-Mouse IgG. Here, the vertical stripes, where MouseIgG is bound, are fluorescent while the horizontal areas do notfluoresce appreciably. Finally, FIG. 10c shows the region treated withboth secondary antibodies (fluorescein-anti-Rabbit and anti-Mouse). Inthis case both the vertical and horizontal stripes fluoresce, indicatinga high surface density fluorescein and, therefore, of the secondaryantibodies. This experiment demonstrates the spatially addressableimmobilization of two different antibodies on the same surface.

What is claimed is:
 1. A method for forming predefined regions on asurface of a solid support, the predefined regions capable ofselectively immobilizing an anti-ligand, the method comprising:a)attaching to the surface a caged biotin analog having a low affinity for(i) the anti-ligand or (ii) a specific binding substance capable ofbinding an anti-ligand, said caged biotin analog comprising aphotolabile chemical protecting group, which chemical protecting groupis removable by irradiation to convert said caged biotin analog to abiotin analog capable of non-covalently immobilizing (i) the anti-ligandor (ii) the specific binding substance by an interaction having anaffinity constant of 107 or stronger; and b) selectively irradiating apredefined region of the surface to convert the caged biotin analogs inthe predefined region to the biotin analog capable of non-covalentlyimmobilizing (i) the anti-liqand or (ii) the specific binding substance.2. The method of claim 1 wherein the caged biotin analog comprises theformula: ##STR32## wherein X and Z are selected from the groupconsisting of a) hydrogen,b) oxycarbonyls of lower alkyl aryl and benzylgroups, c) a nitroveratryloxycarbonyl group, d) anitropiperonyloxycarbonyl group where Z is hydrogen and R isp-nitrophenol formate, e) ##STR33## wherein R₁ and R₂ are hydrogen,lower alkyl, aryl, benzyl, halogen, alkoxyl, thiol, thioether, amino,nitro, carboxyl, formate, sulfonate, formamido or phosphido groups, f)##STR34## wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl,pyrenyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino nitro,carboxyl, formate, formamido, or phosphido groups and R₃ and R₄ arehydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol,thioether, amino, nitro, carboxyl, formate, formamido or phosphidogroups; g) ##STR35## wherein R₁ and R₂ are hydrogen, lower alkyl, arylbenzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro,carboxyl, formate, formamido or phosphido groups, and h) ##STR36##wherein R₁, R₂, R₃ and R₄ are hydrogen, lower alky, aryl, benzyl,halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl,formate, formamido or phosphido groups; provided that when X ishydrogen, Z is not hydrogen or methyloxycarbonyl and provided that whenZ is hydrogen, X is not hydrogen or methyloxycarbonyl;R is selected fromthe group consisting of hydrogen, lower alkyl, aryl, lower alkylformate, aryl formate, methyl formate, p-nitrophenol formate, formamide,N-alkylformamide, N-succinimidyl, hydroxyl, alkoxyl, thiol, thioether,disulfide, hydrazide and amine groups and provided that when X or Z ismethyloxycarbonyl, R is not methyl formate; U is O, S or NH; Y isselected from the group consisting of sulfur, oxygen, methylene,carbonyl, sulfinyl and sulfonyl groups, or Y represents two hydrogenatoms attached to the respective carbons; n=0-7; or acid salts of thecompound.
 3. A method as in claim 1 wherein the binding member isattached to the surface by a crosslinking group.
 4. A method as in claim1 wherein the surface is silica or glass.
 5. A method for attaching ananti-ligand to a surface comprising the steps of:a) linking to thesurface caged biotin analogs having a relatively low affinity foravidin; b) irradiating caged biotin analogs on predefined regions of thesurface to form biotin analogs having high binding affinities foravidin; c) reacting the biotin analogs with either an avidin analog anda biotinylated anti-ligand or an anti-ligand attached to an avidinanalog.
 6. The method of claim 5 wherein the caged biotin analog has theformula ##STR37## wherein X is selected from the group consisting of a)oxycarbonyls of lower alkyl, aryl and benzyl groups, provided that X isnot methyl oxycarbonyl;b) a nitroveratryloxycarbonyl group, c) anitropiperonyloxycarbonyl group, and R is p-nitrophenol d) ##STR38##wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, halogen,alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, sulfonate,formamido or phosphido groups, e) ##STR39## wherein R₁ and R₂ arehydrogen, lower alkyl, aryl, benzyl, pyrenyl, halogen, hydroxyl,alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido,or phosphido groups and R₃ and R₄ are hydrogen, lower alkyl, aryl,benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro,carboxyl, formate, formamido or phosphido groups;R is selected from thegroup consisting of hydrogen, lower alkyl, aryl, benzyl andN-succinimidyl; or acid addition salts of the compound.
 7. A method asin claim 6 wherein the biotin analog has the formula: ##STR40## whereinW is a surface or a crosslinking group attached to a surface.
 8. Themethod of claim 5, wherein the biotin analog and avidin analog have anaffinity constant (K_(a)) of about 10¹⁵ M⁻¹.
 9. The method of claim 5wherein the anti-ligand is attached to the avidin analog by abifunctional crosslinking group.
 10. A method as in claim 5 wherein theanti-ligand is a monoclonal antibody.
 11. A method as in claim 5 whereinthe surface is glass or silica.
 12. A method for attaching a pluralityof anti-ligands having different ligand binding specificities topredefined regions on a surface, said method comprising the steps of:a)attaching to the surface caged biotin analogs having a low affinity forthe anti-ligand or specific binding substances, said caged biotinanalogs comprising a photolabile chemical protecting group; b)irradiating the caged biotin analogs on a first predefined region of thesurface, thereby forming biotin analogs on the first predefined regionhaving an affinity constant of 10⁷ or stronger for an anti-ligand or aspecific binding substance; c) reacting the biotin analogs on the firstpredefined region with either (i)(a) the specific binding substance anda first anti-ligand or (b) a first anti-ligand attached to a specificbinding substance, to form a biotin analog-specific bindingsubstance-anti-ligand complex; or (ii) a first anti-ligand to form abiotin analog-anti-ligand complex; d) washing the surface to removeunbound specific binding substance or anti-ligand; e) repeating steps(b-d) on a different region of the surface with a different anti-ligand.13. The method of claim 12 wherein the anti-ligand is an immunoglobulin.14. A method of screening a plurality of anti-ligands localized onpredefined regions of a surface for affinity for ligands in solutioncomprising:a) attaching to the surface caged biotin analogs having a lowaffinity for the anti-ligand or specific binding substances, said cagedbiotin analogs comprising a photolabile chemical protecting group; b)irradiating the caged biotin analogs on a first predefined region of thesurface, thereby forming biotin analogs on the first predefined regionhaving an affinity constant of 10⁷ or stronger for an anti-ligand or aspecific binding substance; c) reacting the biotin analogs on the firstpredefined region with either (i)(a) the specific binding substance anda first anti-ligand or (b) a first anti-ligand attached to a specificbinding substance, to form a biotin analog-specific bindingsubstance-anti-ligand complex; or (ii) a first anti-ligand to form abiotin analog-anti-ligand complex; d) washing the surface to removeunbound specific binding substance or anti-ligand; e) repeating steps(b-d) on a different region of the surface with a different anti-ligandto create a surface having a plurality of anti-ligands non-covalentlybound on predefined regions of the surface; f) exposing the surface fromstep (e) to a solution containing at least one marked ligand; g) washingthe surface free of unbound ligand; and h) identifying ligands bindingto anti-ligands by determining regions on the surface where markers arelocated.
 15. A method of performing a competitive assay of a ligand insolution by measuring the ligand's affinity for anti-ligands immobilizedon predefined regions of a surface by the steps of:a) attaching to thesurface caged biotin analogs having a low affinity for the anti-ligandor specific binding substances, said caged biotin analogs comprising aphotolabile chemical protecting group; b) irradiating the caged biotinanalogs on a first predefined region of the surface, thereby formingbiotin analogs on the first predefined region having an affinityconstant of 10⁷ or stronger for an anti-ligand or a specific bindingsubstance; c) reacting the biotin analogs on the first predefined regionwith either (i)(a) the specific binding substance and a firstanti-ligand or (b) a first anti-ligand attached to a specific bindingsubstance, to form a biotin analog-specific bindingsubstance-anti-ligand complex; or (ii) a first anti-ligand to form abiotin analog-anti-ligand complex; d) washing the surface to removeunbound specific binding substance or anti-ligand; e) repeating steps(b-d) on a different region of the surface with a different anti-ligandto create a surface having a plurality of anti-ligands non-covalentlybound on predefined regions of the surface; f) incubating a solutioncontaining at one or more marked ligands and unknown ligands with thesurface of step (e); g) washing the surface free of unbound ligand; andh) measuring an amount of marked ligand remaining on the predefinedregions of the surface.
 16. The method of claim 15, wherein step (f)comprises first binding unknown ligands to anti-ligands on the surfaceand then incubating a solution containing one or more marked ligandswith the surface.